Ribosomal protein synthesis is a fundamental biological process whose accuracy is critical for the health and well being of all cells. There is clear evidence that ribosomes contain an "exit" or "E" site for deacylated tRNA and that tRNA passes through it after completing its translational role. Theory suggests that this site is important for maintaining the translational reading frame and helping to ensure accurate aminoacyl-tRNA selection at the ribosomal A site. However, these hypotheses have not been adequately tested experimentally. Our current, funded work provides strong evidence that E-site tRNA helps to hold the reading frame. Aim 1 of this renewal proposal is to further test this model using a novel experimental system that is not dependent on the same assumptions used in current work. The work will determine whether E-site codon:anticodon duplex stability is related to the ability to hold the reading frame. The other two aims are to determine whether E-site tRNA affects aminoacyl- tRNA selection at the A site. Together, these experiments will provide extensive tests of the hypotheses regarding functional roles of the E site and lead to a better understanding of a biochemical process that is critical for the health and well-being of all cells. Finally, this work contributes to the research training of promising biomedical students. The current grant has trained 2 MS students and 7 BS students. So far, it has produced two papers in a top journal (RNA), and another paper is in progress. The renewal should be equally productive, having three Specific Aims, each of which should produce at least one publication. PUBLIC HEALTH RELEVANCE: The proposed work will meet all three goals of the NIH AREA program. First, it will support meritorious research that will advance understanding of biological protein synthesis, which is a fundamental genetic process that is critical for the health and well- being of all organisms. Second, this work will contribute to the research environment at Wake Forest University. Finally, it will contribute to the research training of BS- and MS-level students who are destined for biomedical careers.